Neuraminidase (also known as sialidase, acylneuraminyl hydrolase) is an enzyme common among animals and a number of microorganisms. It is a glycohydrolase that cleaves terminal alpha-ketosidically linked sialic acids from glycoproteins, glycolipids and oligosaccharides. Many of the microorganisms containing neuraminidase are pathogenic to man and other animals including fowl, horses, swine and seals. These pathogenic viruses include influenza.
Influenza is typically transmitted through aerosols as a result of coughing and sneezing by those infected. The virus can also be contracted by exposure to bird droppings, saliva, nasal secretions, feces and blood. The 80-120 nm viral particles consist of an outer envelope and a central core containing the RNA genome, along with various packaging proteins. The influenza genome is not a single piece of RNA, but instead is contained on 8 separate strands of negative-sense RNA, which together encode the 11 genes necessary for viral replication: hemagglutinin, neuraminidase, nucleoprotein, M1, M2, NS1, NS2(NEP), PA, PB1, PB1-F2 and PB2.
Hemagglutinin and neuraminidase are glycoproteins that exist on the outside of the viral particle. During the infection of host cells, the hemagglutinin protein binds to sialic acid residues on the surface of epithelial cells located in the nose, throat, and lungs. The hemagglutinin is subsequently cleaved by host proteases, triggering importation of the viral particle into the host cell by endocytosis. Once inside the cell, the M2 ion channel transports protons from the acidic endosomal fluid into the core of the virus. This drop in internal pH triggers disassembly of the core and release of the viral RNA.
The negative-sense RNA is then transported into the host cell's nucleus, where it is transcribed to the corresponding positive-sense RNA before being exported to the cytoplasm and translated into viral proteins. These are assembled with negative-sense RNA into viral progeny that remain attached to the host cell via hemagglutinin-sialic acid interactions. Finally, the neuraminidase enzyme cleaves sialic acid from the host cell, allowing the newly formed viral particles to infect neighbouring cells.
Because of the absence of RNA proofreading enzymes, RNA transcription results in an error about once every 10,000 nucleotides. Since this is roughly the length of the total RNA present in the influenza genome, evolution is very rapid. Moreover, the separation of the genome into eight separate lengths of RNA permits shuffling of genetic sequences between viruses, if more than one strain of influenza infects a single cell. Together, these mechanisms of “antigenic drift” and “antigenic shift” lead to rapid evasion of established drug or vaccine protocols.
While the influenza virus mostly resides in the lungs, recent evidence that the swine flu strain of H1N1 can penetrate to the gut in animal models raises concerns that drugs with low systemic availability (e.g. zanamivir) will promote reservoirs of the virus elsewhere in the body. These systemic viral reservoirs would be exposed to relatively low doses of the drug over time, providing ideal conditions to evolve further resistance.
Treatment of influenza infections relies on two classes of molecules. The first class includes the molecules amantadine and rimantadine, and works by blocking the M2 proton channel. However, resistance to amantadine and rimantadine is now widespread.
The second class of drugs targets viral neuraminidase (also called sialidase). Zanamivir (Relenza™) was developed in 1989 as a structural mimic of the boat-shaped sialic acid-hydrolysis transition structure, and proved effective in limiting viral replication. However, the large number of heteroatoms within zanamivir's structure limits its oral bioavailability. As a result, it must be administered by inhalation, and has therefore seen somewhat limited clinical use.
Oseltamivir (Tamiflu™) is a second-generation neuraminidase inhibitor developed by Gilead Sciences with substantially improved oral bioavailability. This molecule (marketed by Roche) dominates the influenza market, with sales of about $1 billion per year. Oseltamivir is a prodrug, which is hydrolyzed in the liver to form the biologically active carboxylate. The drug is made in a lengthy synthesis from (−)-shikimic acid, a natural product isolated from the Chinese star anise. Frequent global shortfalls in shikimic acid production threaten Roche's ability to provide large quantities of oseltamivir in response to influenza pandemics. Several alternative syntheses by prominent synthetic groups have appeared in the literature over the past few years, but so far none have been commercialized.
In an attempt to access neuraminidase inhibitors with a core structure distinct from oseltamivir, BioCryst pharmaceuticals developed the substituted cyclopentane peramivir (BCX-1812). This structure, containing a β-hydroxy acid function, is ten-fold more potent than oseltamivir possibly due in part to the interaction of the hydroxyl group with the aspartic acid residue (Asp151) present in the active site to recognize the α-hydroxyl group in sialic acid. Although originally intended as an oral antiviral agent, peramivir displayed poor bioavailability in early trials, and is now being studied in formulations suitable for intravenous and intramuscular injection. Other anti-influenza drugs are also in development, but these likewise suffer various disadvantages.
Because mutations in a key enzyme like neuraminidase are often toxic to the organism, resistance to oseltamivir or zanamivir was once thought to be less problematic than resistance to amantadine. Indeed, data prior to the 2007/2008 flu season showed resistance levels of ˜1%, with somewhat higher levels in children. Resistance increased dramatically in 2007, however, with oseltamivir-resistant H1N1 strains detected in the United States (10.9%), Canada (26%), Europe (25%) and Hong Kong (12%). Even more alarming, data from the first half of the 2008/2009 flu season (prior to the emergence of the swine flu H1N1 strain) showed that nearly all circulating cases of H1N1 influenza A were resistant to oseltamivir.
Both oseltamivir and peramivir were designed on the principle that the polar sidechain of sialic acid (and thus zanamivir) could be replaced by a large, lipophilic alkyl group. While this led to very active inhibitors (oseltamivir is effective at around 1 nM; peramivir is roughly ten-fold more potent), it provides an obvious mechanism for drug resistance. Indeed, influenza strains containing group 1 neuraminidases (N1, N4, N5, N8) are susceptible to a second-shell mutation wherein histidine-274 of the enzyme is mutated to a tyrosine. This mutation results in the reorganization of a nearby glutamic acid residue (Glu276) such that it projects into the active site of the enzyme, where it suffers unfavourable interactions with the lipophilic alkyl group. The H274Y mutation is responsible for the majority of drug resistance described above, conferring resistance to both oseltamivir and peramivir. While strains of influenza expressing these variants remain susceptible to zanamivir (the polar sidechain engages in hydrogen-bonding with Glu276), the lack of oral bioavailability for this drug makes it a less desirable therapeutic.
Moreover, other neuraminidase mutations confer resistance to zanamivir. In addition to several zanamivir-resistant mutants generated in vitro (influenza A N2: E119G/D/A, R292K; influenza A N9: E119G, R292K; influenza B: E119G/D), a recent sampling of Australian and South East Asian influenza A H1N1 viruses revealed a novel mutation (Q136K) which caused a 300-fold reduction in zanamivir susceptibility, as well as a 70-fold reduction in peramivir susceptibility. An earlier zanamivir-resistant strain of influenza B (containing the R152K mutation) was isolated from an immunocompromised patient undergoing prolonged zanamivir treatment.
To effectively combat emerging flu strains and minimize the potential for resistance, novel therapeutic platforms that are effective against the H274Y variant and are sufficiently “plastic” to overcome new polymorphisms are essential.